Engineered CaM2 modulates nuclear calcium oscillation and enhances legume root nodule symbiosis

Significance Oscillations in intracellular calcium concentration play an essential role in the regulation of multiple cellular processes. In plants capable of root endosymbiosis with nitrogen-fixing bacteria and/or arbuscular mycorrhizal fungi, nuclear localized calcium oscillations are essential to transduce the microbial signal. Although the ion channels required to generate the nuclear localized calcium oscillations have been identified, their mechanisms of regulation are unknown. Here, we combined proteomics and engineering approaches to demonstrate that the calcium-bound form of the calmodulin 2 (CaM2) associates with CYCLIC NUCLEOTIDE GATED CHANNEL 15 (CNGC15s), closing the channels and providing the negative feedback to sustain the oscillatory mechanism. We further unraveled that the engineered CaM2 accelerates early endosymbioses and enhanced root nodule symbiosis but not arbuscular mycorrhization.

Plant material. The stable R108/p35S:CNGC15a:Myc:YFP N :T35S line was generated using Agrobacterium tumefaciens AGL1 carrying the recombinant binary vector pB7WG2::CNGC15a:MYC:YFP N according to (10) with the following modifications. AGL1 was used at OD600=0.6 and no acetosyringone was added. Incubation time with AGL1 was 45 min. Leaf explants were prepared by cutting perpendicular to the main vein. Co-cultivation of leaf explants was carried out with their abaxial surface in contact with the medium. The coding region of CNGC15a in frame with the MYC:YFP N was generated by overlapping PCR using the primers listed in the Dataset S2 as described previously (3). MYC:YFP N was amplified from pDEST-GW SCYNE (11). The sequence CNGC15a:MYC:YFP N was cloned into the pB7WG2 (12) via BP/LR gateway cloning.
1.6 Da, charge states 2-5, threshold 1.9e 4 , CE = 30, AGC target 1.9e 4 , max. inject time 35 ms, dynamic exclusion 1 count, 15 s exclusion, exclusion mass window ±5 ppm]. Peaklists were generated with MaxQuant 1.6.17.0 (16) in LFQ mode using the Medicago protein sequence database [Medicago truncatula, BioProject 10791, (57,585 entries)] and the MaxQuant contaminants database (245 entries). The quantitative LFQ results from MaxQuant with default parameters were used together with search results from an in-house Mascot Server 2.4.1 (Matrixscience, London, UK) on the same databases. A precursor tolerance of 6 ppm and a fragment tolerance of 0.6 Da was used. The enzyme was set to trypsin/P with a maximum of 2 allowed missed cleavages; oxidation (M), acetylation (protein N-term) was set as variable modifications; carbamidomethylation (C) as fixed modification. The Mascot search results were combined into Scaffold 4 (www.proteomesoftware.com) using identification probabilities of 99% for proteins and 95% for peptides.  Fig. 16). Two-weeks old transformed roots were analyzed for yellow fluorescent protein (YFP) and mCherry fluorescence using the confocal laser scanning microscope Zeiss LSM780, (YFP: excitation 514nm, emission imaged between 520 and 580nm; mCherry: excitation 587nm, emission imaged between 600 and 620nm). Three biological replicates were analyzed for each construct.
Protein expression and purification. The coding sequence of CaM2, CaM2 R91A and the C-terminal sequence of CNGC15a, CNGC15b and CNGC15c were cloned into the expression vector pOPIN-M (17) by In-Fusion TM reaction (Clontech-Takara). All sequences were PCR-amplified from the yeast two hybrid pBD and pAD clones generated in this study using the primers listed in Dataset S2. The resulting constructs were transformed into Escherichia coli Rosetta™ (DE3) pLysS™ (18), and the expression of the N-terminal His6-MBP-3C tagged proteins were induced by 1 mM isopropylthio-β-D-1galactopyranoside (IPTG) when the culture reached an OD600 of 0.5 at 30 °C. The IPTG-induced cultures were incubated at 16 °C for 14h before collecting the cells by centrifugation at 5663 x g for 10 min and resuspending in 100 mL (ratio: 1/80; volume of buffer/volume of cell culture) of 50 mM Tris-HCl (pH 7.5), 500 mM NaCl, 50 mM glycine, 5% (v/v) glycerol, 20 mM imidazole supplemented with EDTA-free protease inhibitor tablets (Roche cOmplete, EDTA free). Cells were sonicated and following centrifugation at 38,724 x g for 45 min at 4°C, the N-terminal His6-MBP-3C tagged proteins present in the lysate were purified via immobilized metal affinity chromatography using a 5 mL Ni 2+ -NTA column (GE Healthcare), followed by gel filtration on a Superdex™ 200 16/60 column (GE Healthcare). For BLItz and ITC, the MBP tag was cleaved from His6-MBP-3C: CaM2 and His6-MBP-3C: CaM2 R91A , by 3C protease (1:100 ratio of protein:protease) treatment at 4 °C for 16 hours. The cleaved proteins were purified using tandem Ni 2+ -NTA and MBP Trap HP columns (GE Healthcare), followed by gel filtration on a Superdex™ 200 16/60 for final purification and buffer exchange with 20 mM HEPES pH 7.5, 150 mM NaCl buffer. Recombinant protein purity was assessed by SDS-PAGE gel (Sup. Fig. 17).
Biolayer interferometry. Biolayer interferometry (BLItz) was performed to monitor interactions of CaM2 or CaM2 R91A with the C-terminal domain of CNGC15s using BLItz instrument (FortéBio, Menlo Park, CA) and Ni-NTA Biosensors (FortéBio) at room temperature. The Ni-NTA biosensor was hydrated in reaction buffer (20 mM HEPES pH 7.5, 150 mM NaCl) for 10 min before each run. For each run, a baseline was established in the reaction buffer (20 mM HEPES pH 7.5, 150 mM NaCl) for 30 s, then 50 µM of His6-MBP:CNGC15a, His6-MBP:CNGC15b or His6-MBP:CNGC15c was loaded onto the Ni-NTA biosensor tip for 2 min. A baseline was re-established in the reaction buffer (20 mM HEPES pH 7.5, 150 mM NaCl) for 45 s to wash the unbound CNGC15s, followed by a control run in the presence of CaCl2 (at concentration indicated) with an association step of 20s followed by a dissociation step of 20s. The CaM2 binding to CNGC15s was tested using purified CaM2 at the concentration indicated, in absence or presence of CaCl2, with an association step of 20s followed by a dissociation step of 20s.
To calculate the Kinetics parameters (Kd, kon, koff, R 2 ), the corresponding control runs were subtracted, and the final values were determined using a 1:1 Binding Model using BLItz Pro software v1.2 (ForteBio). The replicates were performed with proteins from various number of independent expressions and purifications as indicated.
Isothermal Titration Calorimetry. Isothermal Titration Calorimetry (ITC) was carried out using a MicroCal PEAQ-ITC (Malvern Panalytical). To test the interaction of CaM2 and CaM2 R91A with calcium, the purified proteins were loaded at the final concentration of 100 µM in reaction buffer (20 mM HEPES pH 7.5, 150 mM NaCl). CaM2 and CaM2 R91A were titrated in the reaction buffer with 5 mM CaCl2. Titration was scheduled with 18 consecutive injections of 2 μL of reaction buffer with 5 mM CaCl2, with a 150 s interval between injections. To test the interaction of CaM2 and CaM2 R91A in presence of 5 mM CaCl2 with the IQ domains of CNGC15b and CNGC15c, the custom peptides AACFIQVAWRRTIQEKKG for CNGC15b, and AACFIQAAWRRHKKRKEA for CNGC15c, were synthesized (Merck). For each assay, 10 µM of peptide diluted in the reaction buffer (20 mM HEPES pH 7.5, 150 mM NaCl, 5 mM CaCl2) was titrated through 18 consecutive injections of 2 µL of 150 µM of CaM2 or CaM2 R91A in 20 mM HEPES pH 7.5, 150 mM NaCl and 5 mM CaCl2. Data acquisition and analysis were performed using MicroCal PEAQ-ITC Software (Malvern Panalytical). The replicates were performed with CaMs purified from two independent proteins expressions and purifications.

Structural homology modelling and prediction of calmodulin mutant variant stability and flexibility.
Homology models of CaM2 wild type and mutant were generated by Swiss Model using PDB 5a2h AtCaM7 as a template. The molecule stability and flexibility of the CaM2 mutants was predicted by submitting the model to the DynaMut server (19) (biosig.unimelb.edu.au/dynamut/). Figures were produced in CCP4mg (20).

Calcium oscillation analyses.
Calcium oscillation measurements were performed using a Nikon ECLIPSE FN1 equipped with an emission image splitter (OptoSplit II, Cairn Research) and an electron multiplying cooled charge coupled (Rolera TM Thunder EMCDD) camera (QImaging). ECFP was excited using light emitting diode (OptoLED, Cairn) at 436±20 nm and emitted fluorescence detected at 535±30 nm (cpVenus) and 480±40 nm (ECFP). Images were collected in 3s intervals for 1h30 using MetaFluor software. M. truncatula R108 roots expressing NLS:YC3.6, NLS:YC3.6-CaM2 or NLS:YC3.6-CaM2 R91A , and M. truncatula R108::YC3.6 roots expressing the empty pK7GWIWG2D(II)R or the pK7GWIWG2D(II)R::RNAiCaMs were generated as described above. 2 weeks old transformed plants were placed in a chamber made on a 48x64 mm coverglasses (Solmedia) using high-vacuum grease (Dow Corning GMBH). The chamber was filled with 2 mL of Buffered Nodulation Medium (BNM) (22). Only the root was covered with a coverslip to leave space for Nod factor application at a final concentration of 10 -8 M. Nod factor was produced as previously described (23). Calcium imaging was performed on 2 cm long roots and on the root hair of the induction zone. To obtain a high quality of calcium recording, a maximum of two nuclei were imaged per transformed plants. Calcium oscillation traces were analyzed using GNU Octave v6.1.0 with the nan v3.5.0 and io v2.6.3 packages. The script developed to analyze the frequency, amplitude, rise time and fall time of the calcium spikes is included in Supplemental Dataset 3.

Arbuscular mycorrhiza, nodulation and infection assays.
M. truncatula R108 plants which regenerated roots expressing NLS:YC3.6, NLS:YC3.6-CaM2 or NLS:YC3.6-CaM2 R91A were selected by fluorescence microscopy and grown in controlled environment rooms at 22 °C (80% humidity, 16 h photoperiod, 300 μmol m -2 s -1 light intensity). To monitor AM colonization, plants were grown in Terragreen/Sand (Oil-Dri Company, Wisbech, UK) and inoculated with R. irregularis (Endorize; Agrauxine, France) to the ratio 5:5:1 (Terragreen/Sand/Spores). Colonization was monitored over time in the wild type R108:: NLS:YC3.6 control, and colonization of the assays was analyzed when the wild type reach 10%, 30% and 50% of arbuscules development, named early, mid and late stages of colonization, respectively. Early, mid and late stages of colonization correspond to 20-, 36-, and 50days post inoculation, respectively. The fungal structures were stained in acidic ink as follows; roots were cleared in 10% KOH for 15 min at 95°C, washed 3 times in water and subsequently stained in acidic ink (5% ink, 5% acetic acid) for 4 min at 95°C. The AM root length colonization was quantified using the grid intersect method (24). For nodulation assays, plants were grown in Terragreen/Sand (Oil-Dri Company, Wisbech, UK) to a ratio (1:1) for 7 days and then inoculated with S. meliloti 2011 (OD600=0.01). Nodulation was scored after inoculation as indicated. Infection assays were performed as previously described (25).         Upper panel: Representation of the protein sequence of the Medicago truncatula MCA8 and its Arabidopsis thaliana (At) homolog ECA1 and ECA4. MCA8 belong to the calcium ATPase type IIA which is not regulated by calmodulin. MCA8 shares 78.2% and 78% identity with ECA1 and ECA4, respectively (LALIGN). The calcium ATPase type IIB, ACA4, regulated by calmodulin is represented. Bottom panel: alignment of the N terminus of MCA8 protein sequence with AtECA1, AtECA4 and AtACA4. The calmodulin binding domain (CAMBD) and the transmembrane domain (TM) are highlighted in black and yellow, respectively.  (A) Four parameters characterise the calcium oscillation pattern; the duration of the upward slope of each spike, the amplitude of each spike, the duration of the downward slope of each spike, and the frequency of the calcium oscillation. (B-D) Overexpressing CaM2 R91A which has an increase affinity for CNGC15s is used to unravel the function of CaM2. We hypothesise that (B) if holo-CaM2 closes CNGC15s, holo-CaM2 R91A will decrease the duration and the spike's downward slope and increase frequency of the oscillation, (C) if holo-CaM2 opens CNGC15s, holo-CaM2 R91A will decrease the duration and the spike's upward slope and increase frequency of the oscillation, and (D) if holo-CaM2 is involved in terminating the calcium oscillation, premature arrest of the calcium oscillation will be observed.    (A) Upon a yet unknown activation mechanism (1) of either DMI1 or CNGC15, both ion channels undergo a structural change, putatively unlocking CNGC15 from DMI1. Calcium leak through CNGC15 is predicted to positively feedback on DMI1, increasing its counter-balance flux, and by consequence the calcium release via CNGC15. (2) Holo-CaM2 binds CNGC15 and close CNGC15s whereas MCA8 pumps calcium back to the nuclear envelope lumen. (3) Holo-CaM2 binds upon release of calcium in the nucleoplasm. Each calcium release raises the nucleoplasmic calcium level above 700 nM. Holo-CaM2 binding to CNGC15s is transient and once holo-CaM2 is released from CNGC15, the cycle repeats itself as both ion channels are in an active state. (5) A yet unknown mechanism will stop the nuclear calcium oscillation. (B) CaM2 R91A has higher affinity and association rate (K on ) than CaM2 to CNGC15, and thus outcompete CaM2 in planta leading to accelerated negative feedback (2), which is visualized by a reduction of the duration of the downward slope of each calcium spike and an increase of the calcium oscillation frequency. INE: Inner nuclear envelope. Figure created with BioRender.com  Corresponding amino acid (inluding Methionine as position 1) ΔΔG Residue number in the interlobe linker with low frequency of conservation and exposed to the solvant (including Methionine as position 0)